The invention relates to a protease for activating the blood clotting factor VII, to a process for isolating it, detecting it and inactivating it, and to medicinal preparations which comprise this protease.
The blood clotting system comprises two different, cascade-like pathways for activating clotting factors which are present in the plasma. The intrinsic or the entrinsic pathway is preferentially used for initiating clotting, depending on the triggering mechanism.
When a tissue is damaged, thromboplastin (tissue factor, TF with phospholipids) is exposed by the affected cells as the starter of the extrinsic clotting pathway. The membrane-located thromboplastin can bind both clotting factor VII (FVII) and circulating, activated FVII (FVIIa). In the presence of calcium ions and lipids, this TF-FVIIa complex leads to the binding of FX, which is converted into its activated form (FXa) by limited proteolysis. FXa in turn leads, by activating prothrombin to form thrombin, to the formation of fibrin and thereby ultimately to closure of the wound.
While the further activation of the thromboplastin-bound FVII initially takes place autocatalytically, in particular, it is supported, after the clotting cascade has been initiated, by FXa and thrombin, in particular, leading to marked reinforcement of the reaction cascade.
The administration of FVIIa or FVIIa-containing concentrates is indicated in certain clinical situations. The so-called FVIII-bypassing activity (FEIBA) of FVIIa is used in patients who are suffering, for example, from hemophilia A and have developed antibodies against FVIII as a consequence of the administration of FVIII. According to presently available findings, FVIIa is well tolerated in this context and, while it does not lead to any tendency to thrombosis, it is suitable for ensuring that clotting takes place to a limited but adequate extent. Recombinant FVIIa is already being used therapeutically and prophylactically. FVII which has been isolated from blood plasma can also be activated and then used. Proteases such as thrombin can be used for this activation; however, these proteases, as such, can themselves strongly activate clotting and lead to the risk of a thrombosis. For this reason, subsequent removal or inactivation of thrombin is necessary and leads to yield losses. As a result of the risk of thrombosis which is associated with it, the use of FXa or FIIa (thrombin) is frequently contraindicated and only indicated in emergencies, e.g. in association with extreme loss of blood and unstaunchable hemorrhages.
FVIIa is found in very low concentrations in the plasma of healthy subjects. Only very little is so far known about the formation and origin of FVIIa which is circulating in the blood. Traces of thromboplastin which has been expressed or released in association with cell destruction might play a role in this context. Although it is known that factor XIIa, for example, can lead to FVII activation under certain conditions, the physiological relevance of this reaction has not yet been clarified.
Surprisingly, a FVII-activating protease, which differs from all the previously known proteases, has now been found in connection with fractionating human plasma and certain prothrombin complex concentrates. Investigations into this protease have shown that it exhibits a particularly high amidolytic activity toward the peptide substrate S2288 (HD-isoleucyl-L-prolyl-L-arginine-pNA) from Chromogenix AB, Sweden. A particular feature of this protease is that the amidolytic activity is efficiently inhibited by aprotinin. Other inhibitors, such as the antithrombin III/heparin complex, are also suitable for the inhibition. On the other hand, its activity is increased by heparin and heparin-related substances such as heparan sulfate or dextran sulfate and calcium ions. Finally, it has been found that this protease is able, in a manner dependent on time and on its concentration, to convert FVII into FVIIa. This reaction, too, is inhibited by aprotinin.
Part of the subject matter of the invention is therefore a protease for activating the blood clotting factor VII, which
a) is inhibited by the presence of aprotinin,
b) is increased in its activity by calcium ions and/or heparin or heparin-related substances, and
c) in SDS-PAGE, on subsequent staining in the non-reduced state, has one or more bands in the molecular weight range from 50 to 75 kDa and kDa in the reduced state has a band at 40 to 55 kDa and one or more bands in the molecular weight range from 10 to 35 kDa.
In the following text, the activated form of the protease is termed xe2x80x9cproteasexe2x80x9d whereas the non-activated form is termed xe2x80x9cproenzymexe2x80x9d.
Further investigations with this protease have shown that, after enriching or isolation, it suffers from a rapid loss of activity, which was observed in a solution containing 20 mM tris, 0.15 M NaCl at a pH of 7.5. The addition of albumin at a concentration of 0.1% was not able to prevent the activity of the protease from decreasing by 50% after one hour at room temperature. On the other hand, very good stabilization of the protease was observed in a solution which was buffered to a pH of 6.5 with 50 mM Na citrate. If no particular stabilizers are added to the protease solution, no, or only slight, losses in activity are observed if the solution is adjusted to a pH of between 4 and 7.2, preferably to a pH of between 5.0 and 7.0. However, it is expedient to add stabilizers to the solution, with suitable stabilizers, apart from citrate, being, in particular, glutamate, amino acids, such as arginine, glycine or lysine, calcium ions and sugars such as glucose, arabinose or mannose in quantities of 1-200 mmol/l, preferably in quantities of 5-100 mmol/l. Efficient stabilization was also achieved by adding glycols such as ethylene glycol or glycerol, with quantities of 5-80% by weight, preferably of 10-60% by weight, being used. The pH of the stabilized solution should then be between the pH values 4-9.
While the novel protease, and also the proenzyme, can be obtained by recombinant DNA methods or by production in e.g. the milk of suitable transgenic animals, they can in particular be obtained by fractionation of blood plasma or of prothrombin complex (PPSB) concentrates. The starting material is then first of all subjected to an anion exchange chromatography, which is followed by an affinity chromatography of the eluate. A heparin which is immobilized on a matrix, or a heparin-related substance such as heparan sulfate or dextran sulfate, is particularly suitable for the affinity chromatography. When such a chromatographic method is used, the novel protease and /or the proenzyme can be selectively bound and then eluted once again using known methods. The use of a spacer is advisable for coupling the ligand to the matrix. A heparin-lysine matrix has been found to be particularly suitable for isolating the novel protease.
In SDS-PAGE with subsequent staining, the protease which has been isolated by this method exhibits, in the non-reduced state, one to several bands which lie closely together in the molecular weight range of 55-75 kDa. Following reduction, one to several bands were observed in the molecular weight range of 15-35 kDa and one band was observed at 40-55 kDa. A further band between 60 and 65 kDa, which after scanning and quantitative evaluation, constituted 5-10% of the total protein, showed that non-activated proenzyme was also present. This result was supported by appropriate investigations using monoclonal antibodies against this protease. It was therefore concluded that the proenzyme of this protease can also be prepared, pasteurized and used by the method according to the invention. Part of the subject matter of the invention is therefore the proenzyme of the protease for activating blood clotting factor VII. The proportion of the proenzyme is indicated by the band between 60 and 65 kDa. Corresponding to the amino acid sequence which constitutes the activation region of the proenzyme, thrombin, kallikrein or FXIIa are, in accordance with their substrate specificities, examples of suitable physiological activators of the proenzyme.
Some of the properties of the novel protease which have been described, namely the fact that it can be isolated from plasma or from prothrombin complex (PPSB) concentrates which are derived from plasma, the inhibition of its amidolytic activity by aprotinin and the described migration behavior in SDS-PAGE, both in the reduced and in the non-reduced states, are reminiscent of a protease which was isolated by Hunfeld et al. (Ann. Hematol. 1997; 74; A87, 113; Ann. Hematol. 1998; 76; A101, P294 and Etscheid et al. Ann. Hematol. 1999, 78: A42) from a PPSB concentrate which was not defined in any more detail. In that case, the preparation was essentially achieved using an aprotinin matrix. As a result of the amidolytic cleavage of certain peptide substrates, the activity was described as being a thrombin-like activity. Hunfeld et al. did not find any influence on global clotting parameters such as prothrombin time, Quick or platelet aggregation.
The N-terminal sequencing of the protease described by Hunfeld et al. shows concordances with a protein whose cDNA was described by Choi-Miura et al. (J. Biochem. 119: 1157-1165 (1996)). In its primary structure, the corresponding protein exhibits homology with an enzyme termed hepatocyte growth factor activating enzyme (HGFA).
When two bands which were isolated from SDS-PAGE under reducing conditions were subjected to N-terminal sequencing, the following concordances were established:
Concordances are also found in other test results such as substrate specificity and the ability of the activity to be inhibited. Despite this, it is still not possible at present to assume with confidence that these proteins are identical. At any rate, the previously investigated, abovementioned proteins have not been reported to possess the property of activating FVII or activating other factors (see below).